The soil microbe Bacillus thuringiensis (B.t.) is a Gram-positive, spore-forming bacterium characterized by parasporal crystalline protein inclusions. These inclusions often appear microscopically as distinctively shaped crystals. The proteins can be highly toxic to pests and specific in their toxic activity. Certain B.t. toxin genes have been isolated and sequenced, and recombinant DNA-based B.t. products have been produced and approved for use. In addition, with the use of genetic engineering techniques, new approaches for delivering B.t. endotoxins to agricultural environments are under development, including the use of plants genetically engineered with endotoxin genes for insect resistance and the use of stabilized intact microbial cells as B.t. endotoxin delivery vehicles (Gaertner, F. H., L. Kim [1988] TIBTECH6:S4-S7). Thus, isolated B.t. endotoxin genes are becoming commercially valuable.
Until the last ten years, commercial use of B.t. pesticides has been largely restricted to a narrow range of lepidopteran (caterpillar) pests. Preparations of the spores and crystals of B. thuringiensis subsp. kurstaki have been used for many years as commercial insecticides for lepidopteran pests. For example, B. thuringiensis var. kurstaki HD-1 produces a crystal called a delta endotoxin which is toxic to the larvae of a number of lepidopteran insects.
In recent years, however, investigators have discovered B.t. pesticides with specificities for a much broader range of pests. For example, other species of B.t., namely israelensis and san diego (a.k.a. B.t. tenebrionis, a.k.a. M-7), have been used commercially to control insects of the orders Diptera and Coleoptera, respectively (Gaertner, F. H. [1989] "Cellular Delivery Systems for Insecticidal Proteins: Living and Non-Living Microorganisms," in Controlled Delivery of Crop Protection Agents, R. M. Wilkins, ed., Taylor and Francis, New York and London, 1990, pp. 245-255). See also Couch, T. L. (1980) "Mosquito Pathogenicity of Bacillus thuringiensis var. israelensis," Developments in Industrial Microbiology 22:61-76; Beegle, C. C., (1978) "Use of Entomogenous Bacteria in Agroecosystems," Developments in Industrial Microbiology 20:97-104. Krieg, A., A. M. Huger, G. A. Langenbruch, W. Schnetter (1983) Z. ang. Ent. 96:500-508, describe a B.t. isolate named Bacillus thuringiensis var. tenebrionis, which is reportedly active against two beetles in the order Coleoptera. These are the Colorado potato beetle, Leptinotarsa decemlineata, and Agelastica alni.
Recently, new subspecies of B.t. have been identified, and genes responsible for active .delta.-endotoxin proteins have been isolated (Hofte, H., H. R. Whiteley [1989] Microbiological Reviews 52(2):242-255). Hofte and Whiteley classified B.t. crystal protein genes into 4 major classes. The classes were CryI (Lepidoptera-specific),CryII (Lepidoptera- and Diptera-specific), CryIII (Coleoptera-specific), and CryIV (Diptera-specific). The discovery of strains specifically toxic to other pests has been reported. (Feitelson, J. S., J. Payne, L. Kim [1992] Bio/Technology 10:271-275).
The cloning and expression of a B.t. crystal protein gene in Escherichia coli has been described in the published literature (Schnepf, H. E., H. R. Whitely [1981] Proc. Natl. Acad. Sci. USA 78:2893-2897). U.S. Pat. No. 4,448,885 and U.S. Pat. No. 4,467,036 both disclose the expression of B.t. crystal protein in E. coli. U.S. Pat. Nos. 4,797,276 and 4,853,331 disclose B. thuringiensis strain san diego (a.k.a. B.t. tenebrionis, a.k.a. M-7) which can be used to control coleopteran pests in various environments. U.S. Pat. No. 4,849,217 discloses B.t. isolates which have activity against the alfalfa weevil. U.S. Pat. No. 5,151,363 and U.S. Pat. No. 4,948,734 disclose certain isolates of B.t. which have activity against nematodes. Certain B.t. isolates have been described that have activity against flies, Australian Patent Publication No. AU-B-27866/84. These isolates are not, however, those isolates disclosed and claimed herein. Many other patents have issued for new B.t. isolates and new uses of B.t. isolates. The discovery of new B.t. isolates and new uses of known B.t. isolates remains an empirical, unpredictable art.
The Calliphoridae family, together with the Sarcophagidae and the Oestridae families, contain the species responsible for the most important myiases of domestic animals and man. Myiasis is the infestation of living animals with the larvae of dipteran flies. Myiasis caused by members of the family Calliphoridae is commonly called "blowfly strike." The "blow" is the laying of the eggs by the fly at or near a strike site. The "strike" is the development of the eggs into maggots and the damage that this development causes at that site. Strikes are classified by the area of the body affected.
Blowfly myiasis primarily affects sheep; however, many other animals may be affected. Major species of blowflies include Lucilia sericata (greenbottles), Phormia terraenovae (blackbottles), Calliphora erythrocephala and C. vomitoria (bluebottles) in Europe. These flies are characterized by the color of the metallic sheen on their body sections. Lucilia cuprina, L. caeser, L. illustris, Phormia regina, Calliphora stygia, C. australis, C. fallax, Chrysomyia albiceps, C. chlorophyga, C. micropogon, and C. rufifacies are major species of blowflies in the tropics and subtropics.
The blowflies that attack sheep fall into two main categories:
(1) Primary flies, which are capable of initiating a strike on living sheep. These include Lucilia and Phormia spp. and some Calliphora spp.
(2) Secondary flies, which cannot initiate a strike, but attack an area already struck or otherwise damaged. They frequently extend the injury, rendering the strike one of great severity. Examples include many Calliphora spp. and, in warmer climates, Chrysomyia spp.
Eggs laid on the wool of sheep by primary flies, under favorable conditions, hatch within 12 hours. The hatched larvae migrate down the wool to the skin where the larvae lacerate the skin with their oral hooks and secrete proteolytic enzymes into the skin to establish the lesion. The larvae feed on the surrounding tissues, grow rapidly, and moult twice before becoming fully mature maggots. The maggots then drop to the ground and develop into mature flies. During the period of larval development, extensive tissue damage occurs, and the strike becomes available for the establishment of secondary infections or, worse, becomes an attractive site in which secondary blowflies may lay their eggs.
The irritation and distress caused by blowfly strikes are extremely debilitating, and sheep can rapidly lose condition. Where death occurs, it is often due to septicaemia. Affected sheep are anorexic, appear dull, and usually stay away from the main flock. Current methods of control are based primarily on the prophylactic treatment of sheep with insecticides. The problems associated with this are the relatively short period spent by the larvae on the sheep, the repeated infestations that occur throughout the season, and the rapidity with which severe damage occurs. Any insecticide used must therefore not only kill the larvae, but persist in the fleece. In this respect, the chlorinated hydrocarbon, dieldrin, proved particularly effective and gave protection for at least 20 weeks. However, this product has been largely withdrawn on safety grounds and replaced mainly by organophosphorus compounds, which have a persistence of 10-16 weeks unless resistance supervenes wherein this period becomes much shorter.
Application of these insecticides is made by plunge dipping or, more rarely in Europe, in a spray race or by jetting. In Europe, the high prevalence of body strike makes whole body protection necessary, and therefore the use of dips is more effective. In practice, an annual dip, usually in June, should give protection for the remainder of the fly season, but a second dipping in August may be necessary in order to ensure complete protection.
The name screw-worm is given to the larvae of certain species of Cochliomyia (syn. Callitroga) including C. hominivorax and C. macellaria, and to that of a single species of Chrysomyia, C. bezziani, which cause screw-worm myiasis in animals and occasionally man. Cochliomyia is found in the New World, while C. bezziani is confined to Africa and Asia.
The bluish-green flies have longitudinal stripes on the thorax and orange-brown eyes (Pl. IX). They occur primarily in tropical areas and lay their eggs on wounds, the larval stages characteristically feeding as a colony and penetrating the tissues creating a large and foul-smelling lesion. C. hominivorax was such a problem in the southern United States that a mass eradication campaign using biological control was undertaken. This involved the release of up to 1,000 male flies, sterilized by irradiation, per square mile. Since the female fly mates only once, control proved very successful except where the flies, which are capable of flying up to 200 miles, migrated from across the Mexican border.
Regular use of chemicals to control unwanted organisms can select for drug resistant strains. This has occurred in many species of economically important pests. Biological control programs circumvent the selection problem of drug resistance and are ecologically favored; however, as illustrated by the use in screw-worm control, can be limited by the biology of the fly. The development of drug resistance and the limitations of biological control programs necessitate a continuing search for new control agents having different modes of action.
At the present time there is a need to have more effective means to control these pests that cause considerable damage to susceptible hosts. Advantageously, such effective means would employ biological agents.